US20190212347A1 - Bio-analytical method for insulin analogues - Google Patents

Bio-analytical method for insulin analogues Download PDF

Info

Publication number
US20190212347A1
US20190212347A1 US16/070,651 US201716070651A US2019212347A1 US 20190212347 A1 US20190212347 A1 US 20190212347A1 US 201716070651 A US201716070651 A US 201716070651A US 2019212347 A1 US2019212347 A1 US 2019212347A1
Authority
US
United States
Prior art keywords
insulin
labelled
analogue
insulin analogue
biological matrix
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/070,651
Other languages
English (en)
Inventor
Madhavan Buddha
Mukesh B. Patale
Anand Khedkar
Ranitendranath Tagore
Sebastian Alastair Mcdonald
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biocon Ltd
Original Assignee
Biocon Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biocon Ltd filed Critical Biocon Ltd
Assigned to BIOCON LIMITED reassignment BIOCON LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BUDDHA, Madhavan, KHEDKAR, ANAND, PATALE, MUKESH B., TAGORE, Ranitendranath, MCDONALD, SEBASTIAN ALASTAIR
Publication of US20190212347A1 publication Critical patent/US20190212347A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4745—Insulin-like growth factor binding protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S930/00—Peptide or protein sequence
    • Y10S930/01—Peptide or protein sequence
    • Y10S930/02—Containing modified or unusual amino acid
    • Y10S930/022—Containing radioactive atom

Definitions

  • the present invention relates to a specific and sensitive bio-analytical method for detection of insulin or insulin analogues in plasma, serum or any other biological fluid. It particularly relates to labelling of biological compound with stable isotopic nitrogen and bio-analytical method using solid phase extraction and liquid chromatography with tandem mass spectrometric detection.
  • proteins based biological drugs it is necessary to detect proteins such as insulin contained in the plasma, serum or any other biological fluid, and measure them quantitatively in order to elucidate the function of the protein, or for testing and screening of the protein, or to measure the systemic exposure of the protein. Therefore, if a more general method for quantifying the amount of insulin with a high sensitivity is established, it becomes possible to estimate drug kinetics during preclinical testing or in clinical trials. Thus, it would be possible to estimate drug kinetics in human at an early stage of drug development.
  • RIA radioimmunoassay
  • ELISA ELISA
  • Attempts to generate antibodies specific to Insulin analogue ‘IN-105’ were not successful, as the immunisation cycles yielded antibodies which showed identical binding to human insulin and IN-105, indicating that the addition of the small alkyl polyethylene glycol (PEG) does not alter the structure significantly enough to differentiate the molecule immunologically.
  • the difference in molecular weight between the two molecules is 217 Daltons due to the alkyl PEG in IN-105.
  • a specific ELISA based estimation assay could not be developed for IN-105.
  • LC/MS/MS instrumentation involve liquid chromatography (LC) for separation of analytes, followed by ionisation chamber where the sample undergoes desolvation and ionisation and the masses are then detected by mass spectrometer.
  • LC liquid chromatography
  • MRM multiple reaction monitoring
  • the human insulin protein is composed of 51 amino acids, and has a molecular mass of 5808 Da. It is a dimer of an A-chain and a B-chain, which are linked together by disulphide bonds.
  • analogues of human insulin are available such as IN-105, Insulin Aspart, Insulin Lispro, and Insulin Glargine. These insulin analogues are closely related to the human insulin structure, and were developed for specific aspects of glycaemic control in terms of fast action (prandial insulin) and long action (basal insulin).
  • a specific LC/MS/MS method could be developed for measurement of intact IN-105 molecule in plasma.
  • Sample preparations could be carried out using offline solid phase extraction of the analyte, followed by online solid phase extraction before analysis of the samples by LC/MS/MS.
  • the object of present invention is to provide a sensitive analytical method for the detection and quantification of insulin or insulin analogues IN-105 in human plasma at the concentration range of 0.20 ng/ml to 50.0 ng/ml.
  • the present invention relates to a bio-analytical method for the detection of insulin or insulin analogues such as IN-105 in human plasma.
  • the method comprises labelling of insulin or insulin analogue by a stable isotope of nitrogen i.e. 15 N. Labelling is attained by stable isotope of nitrogen ( 15 N) by providing labelled nitrogen in growth and fermentation medium. The cells are grown in labelled medium therefore almost all nitrogen atoms in insulin or insulin analogues are substituted with stable isotope 15 N.
  • the labelled nitrogen source used for the method is ammonium sulphate [( 15 NH 4 ) 2 SO 4 ].
  • Another aspect of the present invention relates to determination of intact molecule of the labelled insulin or insulin analogues which is further determined using solid phase extraction and liquid chromatography with mass spectrometric detection.
  • FIG. 1 is flowchart of scheme of overall process
  • FIG. 2 is flowchart of upstream process
  • FIG. 3 is process of conversion of IN-105 precursor into IN-105 through trypsin and carboxypeptidase B treatment.
  • FIG. 4 is chromatography peak for Reagent Blank
  • FIG. 5 is chromatography peak for Double Blank
  • FIG. 6 is chromatography peak for Matrix Blank
  • FIG. 7 is Calibration Standard at 0.200 ng/mL LLOQ (Lower Limit of Quantification)
  • FIG. 8 is Calibration Standard 50.0 ng/mL ULOQ (Upper Limit of Quantification)
  • FIG. 9 is QC 0.200 ng/mL LLOQ
  • FIG. 10 is QC 0.600 ng/mL LoQC
  • FIG. 11 is QC 20.0 ng/mL MeQC
  • FIG. 12 is QC 40.0 ng/mL HiQC
  • FIG. 13 is AUCt v DOSE graph of pharmacokinetic study
  • FIG. 14 is C. v DOSE graph of pharmacokinetic study
  • FIG. 15 is Plot of Mean Plasma Concentration for IN-105 30 mg
  • Present invention relates to bio-analytical method for detection of insulin or insulin analogues using liquid chromatography with tandem mass spectrometry.
  • the present invention relates to a highly specific method for quantification of IN-105 in biological matrix.
  • biological matrix is whole blood, blood serum, blood plasma, urine, fermentation broth or buffer.
  • the present method allows us to determine IN-105 exposure under variety of disease conditions.
  • Method can distinguish IN-105 from co-administered insulin/insulin analogue and endogenous insulin. Due to the use of mass spectrometry, the method can be used to determine different insulin analogues simultaneously as long as there is a difference in the molecular weight.
  • the method can be further developed to additionally determine C-peptide levels along with insulin or its analogues.
  • the method in general provides a useful alternative to immunological methods.
  • FIG. 1 The scheme of the process is depicted in FIG. 1 .
  • IN-105 is a novel ‘rapid acting insulin analogue’ being developed for oral delivery as disclosed and described in U.S. Pat. No. 9,101,596.
  • the molecule has an amino acid sequence identical to that of human insulin, except that it has been conjugated with a short chain alkyl-polyethylene glycol molecule at the ⁇ -amino group of lysine at the 29 th position of the B-chain.
  • IN-105 could be selectively quantified in presence of any human insulin or co-administered insulin like insulin Glargine. This makes the method very useful in determining pharmacokinetic exposure of IN-105 following oral administration as demonstrated from the pharmacokinetic profile obtained from 10 mg dosing in patients with T1D (Type 1 Diabetes).
  • Upstream process involves labelling of insulin or insulin analogues by stable isotopic non-metal elements such as nitrogen ( 15 N), sulphur ( 34 S), carbon ( 13 C), oxygen, or hydrogen.
  • the isotopic nitrogen ( 15 N) source in the culture medium is at least one of ammonium sulphate, Ammonium phosphate, Ammonium hydroxide, Methylamine, Urea;
  • the isotopic carbon ( 13 C) source in the culture medium is at least one of Methanol, Glycerol, Glucose, Sorbitol;
  • isotopic sulphur ( 34 S) source in the culture medium is at least one of Calcium sulphate, Magnesium sulphate, Potassium sulphate; D20 for hydrogen and H 2 O 18 for oxygen labelling.
  • the scheme of the process is depicted in FIG. 2 .
  • Downstream process involves validation method for determining pharmacokinetic exposure in normal and disease plasma from human subjects.
  • the validation study included linearity, accuracy and precision, selectivity and specificity, repeatability and reproducibility.
  • the upstream process begins with seed culture preparation, which involves inoculation of seed flasks from the culture of cell bank prepared by using a recombinant strain, Pichia pastoris .
  • the strain carries a gene which codes for the expression of insulin precursor.
  • the seed flasks were grown over a period to increase the cell mass which will be used as the inoculum to start the production fermenter.
  • Fermentation is carried out in two phases viz. batch phase and fed batch phase.
  • batch phase the fermentation is performed to increase the cell mass using glycerol as carbon source and during fed batch phase methanol is used to induce the secretion of insulin precursor along with 15 N labelled ammonium sulphate as the only nitrogen source during process.
  • the insulin precursor protein with labelled 15 N is released into the supernatant during fermentation.
  • the downstream purification process serves to isolate the insulin precursor from the cell-free supernatant and convert it into a Methoxy-triethyleneglycol-propionyl-N ⁇ B29 recombinant insulin precursor, which through a trypsin and carboxypeptidase B catalysed reaction gets converted into insulin.
  • Multiple HPLC steps are used to resolve close product-related impurities during this process.
  • Multiple chromatography steps are used to resolve product related impurities.
  • the product is crystallized and lyophilized.
  • the product Upon obtaining 15 N labelled product of insulin or insulin analogue, the product was used as internal standard in analytical method for determination of IN-105 in plasma. Analytical method can be used for the determination of insulin or insulin analogues in human plasma or other biological fluids containing K 2 EDTA or K 3 EDTA (either Di-potassium or Tri-potassium EDTA).
  • Samples are prepared using off-line Solid-phase extraction (SPE) extraction, followed by on-line SPE in 96-well format.
  • SPE Solid-phase extraction
  • the method relates to determination of insulin or insulin analogues in human plasma.
  • the method is employed to determine insulin or insulin analogues over the concentration range 0.200 ng/mL to 50.0 ng/mL.
  • Inoculum Flask The procedure for labelling begins with seeding of cell line in inoculum flask (either directly or through seed flask) that contain medium, designed for same as shown in table 1.
  • the three components were mixed in 2000 mL flask and was inoculated with one culture vial. 3.
  • the flask was kept in shaker incubator for incubation at 30° C. ⁇ 1° C. for 24 ⁇ 2 hr.
  • composition of fermentation medium described in table 2-4 Composition of fermentation medium described in table 2-4.
  • Biotin (0.2 g/L) was dissolved in potable water and filter sterilized through 0.2 ⁇ before use.
  • Fermentation medium was then autoclaved at 121° C. for 60 min.
  • DO was first maintained by increasing the RPM manually. When DO was reached to 40%, RPM was increased by 200 in one step up to maximum.
  • DO Dissolved Oxygen
  • Methanol flow rate was checked periodically by using balance (0.8 density factor). Balance reading were recorded continuously.
  • the fermentation was checked periodically for methanol accumulation (1-5 min) during methanol induction phase as mentioned in the table 5.
  • MIP Methanol Induction Phase
  • MIP Age hrs.
  • Methanol Feed Rate g/hrs.
  • the insulin or insulin analogue precursor protein with Labelled 15 N is released into the supernatant during fermentation.
  • pH of final broth is adjusted to pH 2.5 with ortho-phosphoric acid followed by centrifugation to get cell free supernatant which further subjected to the downstream purification process to isolate the insulin or insulin analogue precursor from the cell-free supernatant and convert it into a Methoxy-triethyleneglycol-propionyl-N- ⁇ -B29 recombinant insulin or insulin analogue precursor, which through a trypsin and carboxypeptidase B catalysed reaction gets converted into insulin or insulin analogue.
  • the scheme of the process of conversion of IN-105 precursor is depicted in FIG. 3 .
  • K 2 EDTA or K 3 EDTA Control Human Plasma from patients diagnosed with either type one (T1DM) or type two diabetes (T2DM) were also obtained. Plasma from patients with type 1 and type 2 diabetes was procured from external supplier and was available either Di-potassium EDTA (K 2 EDTA) or Tri-potassium EDTA (K 3 EDTA).
  • K 2 EDTA or K 3 EDTA was obtained from volunteers for use in whole blood stability experiment and in preparation of the matrix used in the assessment of haemolysis (2% whole blood in plasma).
  • Lipaemic plasma was prepared by the addition of intralipid to control human plasma (K 2 EDTA) in a ratio 1:9, was used to assess the impact of lipaemia on the method. Haemolysed (2% whole blood in plasma) plasma was also obtained.
  • Calibration stock solution was prepared by adding 50 ⁇ L of stock solution and diluting it in 450 ⁇ L of diluent (0.1% TFA in acetonitrile:water 50:50 v/v).
  • Quality control (QC) samples were prepared as per table 6 in manner having final concentration of 0.2 (LLOQ QC), 0.6 (LoQC), 20 (MeQC), 40 (HiQC) and 50 (ULOQ QC). These QC samples were stored at ⁇ 20° C. and ⁇ 80° C. for a period of 73 days and were used only for validation purpose.
  • the final calibration standard concentrations were 0.2, 0.4, 1, 5, 10, 20, 45 and 50 ng/mL.
  • nitrogen isotope labelled insulin having purity 96.3% and 96.1% respectively was used as analytical standard and internal standard for further analysis.
  • Glargine (3.64 mg/mL) of 100% purity was used as co-administered standard in the analytical procedure. The stability studies to understand storage stability and solution stability were carried out.
  • IN-105 for stability assessment of IN-105 in whole blood, IN-105 was spiked in whole blood samples at LoQC and HiQC concentrations. The samples were sub-aliquoted and stored at room temperature and on ice. Aliquots of whole blood were taken up to 2 hrs. The samples were processed for analysis by centrifugation at 4000 rpm at 20° C. for 10 minutes. Separated plasma was harvested and stored at ⁇ 80° C. until the time of analysis.
  • Solid phase extraction was carried out using waters Oasis Max (10 mg) SPE plate. Samples were prepared using off-line SPE extraction followed by on-line SPE. Upon obtaining labelled insulin, calibration standard and quality control standards were prepared.
  • the SPE cartridges were conditioned with methanol (300 ⁇ L) and equilibrated with 300 ⁇ L of water. Samples were loaded on to the plate, for ensuring adequate mixing they were aspirated and dispensed. Plate was washed with 500 ⁇ L 1M ammonium acetate in MeCN:water (1:10:90 v/v/v), followed by washing with 500 ⁇ L 1M ammonium acetate in MeCN:water (1:50:50 v/v/v). Packing material was dried using maximum pressure. 0.1% Triton X-100 was added to a fresh 1 mL well plate.
  • Mobile phase A was TFA 0.02% in acetonitrile:water (10:90 v/v); Mobile phase B was TFA 0.02% in acetonitrile:water (90:10 v/v).
  • Initial concentration of mobile phase A was 85% till 30 s, from 30 s to 4 min 30 s, mobile phase A changed from 85% to 60% in a linear fashion, over next 10 s mobile phase A changed to 10% and stayed at 10% till 5 min 30 s.
  • the run time was completed at 6 min 30 s. Temperature of elution was maintained at 50° C. Elution was carried out using 0.5 mL/min flow rate. Injection volume was 500 ⁇ L.
  • wash solvent 1 was 0.01% TFA in acetonitrile:water (10:90 v/v)
  • Wash solvent 2 was 0.01% TFA in methanol:water: Triton-X100 (70:30:0.05 v/v/v)
  • wash solvent 3 was Ammonia (0.5% of 28%) in methanol:water (80:20 v/v).
  • Cartridges were conditioned using 500 ⁇ L methanol at 5 mL/min flow rate. Online cartridges were equilibrated with 500 ⁇ L of (0.1% TFA) in MeCN: water (10:90 v/v). Backflush wash was given using 1 mL of (0.1% TFA) in MeCN:water (10:90 v/v) at 2 ml/min flow rate. Following that cartridges were washed using 1 mL of (0.1% TFA) in MeCN:water (20:80 v/v) at 5 ml/min flow rate.
  • Clamp flush 1 was carried out using 500 ⁇ L of (0.1% TFA) in MeCN:water (10:90 v/v) at 5 mL/min flow rate
  • clamp flush 2 was carried out using (0.1% TFA) in MeCN:water (90:10 v/v) at 5 ml/min flow rate
  • Clamp flush 3 was carried out using 500 ⁇ L of (0.1% TFA) in MeCN:water (10:90 v/v) at 5 ml/min flow rate.
  • the validation study included linearity, accuracy and precision, selectivity and specificity, repeatability and reproducibility.
  • Validation consisted of intra run precision and bias, inter-run precision and bias, dilution integrity, matrix related modification of ionisation, auto-injector carry over, stability in whole blood at room temperature and on ice, freeze thaw stability of plasma over 5 cycles, 24 h room temperature stability of plasma, frozen stability in plasma at ⁇ 20 and ⁇ 80° C. for 73 days, recovery and extract stability at 10° C.
  • the acceptance criteria for mean intra-run and the inter-run precision was less than, or equal to, 15% at all levels, other than at the LLOQ, at which precision had to be better than, or equal to, 20%.
  • the mean intra-run and inter-run bias had to be within ⁇ 15% of the nominal concentration at all levels, other than at the LLOQ, at which bias had to be within ⁇ 20% of the nominal concentration.
  • FIG. 4-12 show the peaks obtained during LC/MS/MS of samples.
  • the method was not affected by inter-individual variability, lipaemia, and haemolysis or by presence of Glargine. The observation infers that method is sufficiently accurate and precise and have sufficient selectivity and reliability that allow determination of insulin in human plasma samples over the examined range.
  • Insulin was found to be stable in plasma when stored at room temperature for up to 24 hours, after five freeze/thaw cycles and after 244 days storage at both ⁇ 20° C. and ⁇ 80° C. Also found to be stable in extracts when stored at 10° C. for approximately 85 hours.
  • Labelled insulin was stable in acetic acid (2%) in methanol:water (30:70 v/v) when stored at ⁇ 20° C. for up to 40 days and when stored at room temperature for up to 24 hours.
  • the eluate from the analytical chromatography column passes into a triple quadrupole mass spectrometer, where an ESI process generates gas-phase ions from the eluate. These gas phase ions were then analysed in MRM mode as follows.
  • the ions pass into the first quadrupole, where ions with m/z values in a narrow range are selected. Ions within this narrow m/z range are allowed to pass through to the second quadrupole.
  • the third quadrupole will be set to select for ions having m/z values characteristic of fragments generated from IN-105 and ISTD. Fragments from other species that happened to have the same intact m/z as IN-105 and ISTD will be eliminated at this stage.
  • the method was considered validated successfully since it passed all the pre-determined criteria in the validation protocol.
  • the validated method was subsequently used for measurement of IN-105 levels in a euglycemic clamp study carried out in patients with Type 1 diabetes.
  • the method for the determination of IN-105 in human plasma has been validated successfully over the concentration range 0.200 ng/ml to 50.0 ng/mL.
  • Plasma concentration data for each patient and treatment was analysed by a non-compartmental method.
  • the area under plasma level curve for AUC0-t was calculated by the trapezoidal rule.
  • the primary pharmacokinetic parameters (mean ⁇ SD) were C max , AUClast, T max and PD parameters were T min , C min , AUClast.
  • Ratios and 90% CIs of geometric means were calculated for PK and PD parameters from mixed effects model with fixed effects for sequence, period and treatment, and patients within sequence as a random effect for log transformed C max and AUC.
  • the maximal plasma concentration of IN-105 as well as the area under the PK curve are linearly correlated significantly (p ⁇ 0.05) with the dose employed (as per table 16 below).
  • the average plasma concentration time profile of IN-105 obtained after administration of IN-105 tablets in volunteers is shown in FIGS. 13-14 .
  • Plots of mean plasma concentration as a function of time after dosing shows the expected time profile of drug concentration in plasma.
  • the plot for the 30 mg/kg dose is shown in FIG. 15 , where a well-defined peak concentration and subsequent clearance is evident.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Endocrinology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US16/070,651 2016-01-23 2017-01-20 Bio-analytical method for insulin analogues Abandoned US20190212347A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
IN201641002615 2016-01-23
IN201641002615 2016-01-23
PCT/IB2017/050303 WO2017125885A1 (en) 2016-01-23 2017-01-20 Bio-analytical method for insulin analogues

Publications (1)

Publication Number Publication Date
US20190212347A1 true US20190212347A1 (en) 2019-07-11

Family

ID=59361626

Family Applications (1)

Application Number Title Priority Date Filing Date
US16/070,651 Abandoned US20190212347A1 (en) 2016-01-23 2017-01-20 Bio-analytical method for insulin analogues

Country Status (8)

Country Link
US (1) US20190212347A1 (de)
EP (1) EP3405785B1 (de)
JP (1) JP6893214B2 (de)
KR (1) KR20180103961A (de)
CN (1) CN108474773A (de)
AU (1) AU2017208935A1 (de)
CA (1) CA3010566A1 (de)
WO (1) WO2017125885A1 (de)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116858978A (zh) * 2023-09-04 2023-10-10 军科正源（北京）药物研究有限责任公司 一种同时检测门冬胰岛素和德谷胰岛素的方法及其血浆样品处理方法
WO2025080523A1 (en) * 2023-10-11 2025-04-17 Quest Diagnostics Investments Llc Methods for the simultaneous detection and quantification of insulin, proinsulin, proinsulin metabolic intermediates, c-peptide, and c-peptide variants by mass spectrometry

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109991351A (zh) * 2019-03-27 2019-07-09 苏州旭辉检测有限公司 一种血液样本中胰岛素类似物的检测方法
WO2025210538A1 (en) 2024-04-02 2025-10-09 Biohaven Therapeutics Ltd. Bifunctional degraders
CN119192319A (zh) * 2024-10-21 2024-12-27 杭州墨梵健康科技有限公司 一种新型索马甜突变体、其制备方法及其应用

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4596207B2 (ja) * 2000-05-15 2010-12-08 味の素株式会社 酵素によるタンパク質の同位体標識法
US20020123055A1 (en) * 2000-08-25 2002-09-05 Estell David A. Mass spectrometric analysis of biopolymers
US20030119109A1 (en) * 2001-07-26 2003-06-26 Van Den Burg Harrold Alfred Efficient 13C/15N double labeling of proteins in a methanol-utilizing strain (Mut+) of pichia pastoris
AU2003268693A1 (en) * 2002-09-30 2004-04-19 Ajinomoto Co., Inc. Method of producing stable isotope-labeled protein
ES2546016T3 (es) * 2005-10-13 2015-09-17 Biocon Limited Procedimiento para la preparación de conjugados de insulina
US20100311176A1 (en) * 2007-05-23 2010-12-09 Applied Biosystems, Llc Method of mass analysis of target molecules in complex mixtures
EP2205972B1 (de) * 2007-10-29 2012-01-04 University Of Georgia Research Foundation, Inc. In-vivo-isotopenkenmarkierungsverfahren für quantitative glycomics
BRPI0905853B1 (pt) * 2008-02-06 2019-03-26 Biocon Limited Processo para a produção de proteínas recombinantes, melhorando a incorporação de fosfato.
CN107064330B (zh) * 2010-12-28 2022-02-25 探索诊断投资公司 通过质谱法定量胰岛素
CN103454433B (zh) * 2012-06-05 2016-08-03 许洋 一种新型检测内外源性胰岛素的免疫质谱试剂盒
US9134319B2 (en) * 2013-03-15 2015-09-15 The Regents Of The University Of California Method for replacing biomarkers of protein kinetics from tissue samples by biomarkers of protein kinetics from body fluids after isotopic labeling in vivo
CN104458890B (zh) * 2014-12-17 2017-03-22 中国计量科学研究院 一种同位素稀释质谱法测定胰岛素含量的方法
US10324082B2 (en) * 2015-03-03 2019-06-18 Quest Diagnostics Investments Llc Methods for quantitation of insulin levels by mass spectrometry

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116858978A (zh) * 2023-09-04 2023-10-10 军科正源（北京）药物研究有限责任公司 一种同时检测门冬胰岛素和德谷胰岛素的方法及其血浆样品处理方法
WO2025080523A1 (en) * 2023-10-11 2025-04-17 Quest Diagnostics Investments Llc Methods for the simultaneous detection and quantification of insulin, proinsulin, proinsulin metabolic intermediates, c-peptide, and c-peptide variants by mass spectrometry

Also Published As

Publication number Publication date
KR20180103961A (ko) 2018-09-19
CN108474773A (zh) 2018-08-31
EP3405785A1 (de) 2018-11-28
CA3010566A1 (en) 2017-07-27
EP3405785B1 (de) 2020-04-29
EP3405785A4 (de) 2019-01-16
WO2017125885A1 (en) 2017-07-27
AU2017208935A1 (en) 2018-08-30
JP2019505786A (ja) 2019-02-28
JP6893214B2 (ja) 2021-06-23

Similar Documents

Publication Publication Date Title
EP3405785B1 (de) Bioanalyseverfahren für insulinanaloga
AU2007340558B2 (en) Method for quantification of peptide and protein
CN104246508B (zh) 多肽产品出厂检验中的杂质定量
US20090246814A1 (en) Reagent for digestion of hemoglobin
Srikanth et al. Correlation of in vitro and in vivo plasma protein binding using ultracentrifugation and UPLC-tandem mass spectrometry
WO2022110569A1 (zh) 一种大鼠血浆中芦荟苦素的lc-ms/ms测定方法
CN109900841B (zh) 一种同时测定血浆中氨基糖苷类抗生素药物浓度的hplc-ms/ms方法
Kuuranne et al. Insulins in equine urine: Qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse‐racing
Oztug et al. An LC-MS/MS-based platform for the quantification of multiple amyloid beta peptides in surrogate cerebrospinal fluid
CN110133169A (zh) 一种采用液质联用检测人血浆中呋塞米的方法及应用
Gao et al. Two validated liquid chromatography–mass spectrometry methods with different pretreatments for the quantification of an anti-CD47 monoclonal antibody in rat and cynomolgus monkey serum compared with an electrochemiluminescence method
CN112326816B (zh) 一种定量检测血清中地舒单抗的液质联用方法
CN119438466A (zh) 特异性抗体免疫磁珠富集血清人胰岛素的质谱检测方法
CN116593621B (zh) 一种利用高效液相色谱-串联质谱联用装置定量分析血浆样本中药物的方法
Zheng et al. Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: a new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics
CN112415114A (zh) 人血浆中缬沙坦和沙库巴曲的浓度的测定方法及其用途
Li et al. Simultaneous determination of omeprazole and domperidone in dog plasma by LC-MS method
US20230366888A1 (en) Use of lc-ms/ms to quantitate protein biomarkers
Sistik et al. First Dimension Trap‐and‐Elute Combined with Second Dimension Reversed‐Phase Liquid Chromatography Separation Using a Two‐Dimensional‐Liquid Chromatography‐Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing
CN116183801A (zh) 检测胰岛素和c肽的液相色谱-质谱方法、试剂盒和系统
Grifnée et al. Development and validation of a highly-sensitive, quantitative LC-MS/MS assay to evaluate plasma oxytocin
CN114624362A (zh) 一种检测血清中晚期糖基化终末产物的试剂盒及其应用
CN114397379A (zh) 一种液质联用测定血浆中奥硝唑浓度的方法
Gervasoni et al. Determination of asymmetric dimethyl arginine in human serum by liquid chromatography-tandem mass spectrometry: clinical application in hypertensive subjects.
Zhang et al. Quantitative analysis of a novel antimicrobial peptide in rat plasma by ultra performance liquid chromatography–tandem mass spectrometry

Legal Events

Date Code Title Description
AS Assignment

Owner name: BIOCON LIMITED, INDIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BUDDHA, MADHAVAN;PATALE, MUKESH B.;KHEDKAR, ANAND;AND OTHERS;SIGNING DATES FROM 20160121 TO 20160122;REEL/FRAME:046887/0054

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO PAY ISSUE FEE